Bacterial gene transcription is a complex, multifactorial process that is driven by the activity of DNA- dependent RNA Polymerase (RNAP). This multi-subunit enzyme consists of: ?, ??, two identical ? subunits and ?. In Gram-positive organisms an additional accessory subunit also exists, known as ?- factor (RpoE). In 40+ years of study, the ? subunit of RNAP has been shown to have wide influence on transcription, and to impact the growth and physiology of a wealth of important organisms. Mutant strains lacking this factor have prolonged lag phases, diminished resistance to extended starvation, and sensitivity to environmental stress. In the context of pathogenic species (including S. aureus, which is the focus of this application), in every organism thus far tested, the deletion of ? has resulted in impaired virulence. In spite of the key role ? plays in bacteria, exactly how it exerts its influence is far from understood. Increasing evidence suggests that its role is mediated by the recognition of specific promoter features of ?-regulated genes. This hypothesis is based on the following lines of evidence: i) There is only a single feature conserved for ? when studied across a wealth of Gram-positive species, an N-terminal Helix-turn-Helix (HTH) domain. To date, all HTH domains studied have a role in recognizing, and binding to, DNA. ii) Multiple studies demonstrate that ? reduces binding of RNAP to intergenic/promoter-less regions of DNA. iii) Multiple groups have shown that ?- and ?-factors exhibit a level of cooperativity, where both elements are required for specific and direct binding of RNAP to promoter regions. iv) The ? subunit has a role in recognizing the initiating nucleotide of transcription. v) Recent work reveals that the ? subunit binds to DNA in the promoter region of the B. subtilis abrB and rrnB1 genes. Collectively, this speaks to the specific and coordinated influence of ? on unique features within the promoters of target genes. Accordingly, we propose to perform the first structure-function relationship study on ? using S. aureus as a model. To achieve this we will: 1. Identify and characterize promoter features that result in ? subunit dependency using cutting edge next-generation sequencing technologies coupled with biochemical and genetic approaches. We will then: 2. Determine the regions of RpoE required for RNAP-binding and promoter recognition by identifying which amino acid residues and/or domains are required to mediate binding to RNAP, and facilitate transcriptional selectivity. Collectively, we will generate data that, for the first time, provides mechanistic understanding to an overlooked component of the Gram-positive transcriptional machinery. This information will deepen our understanding of how the transcription complex interacts with bacterial promoters, and produce findings that could be used for the development of novel anti-virulence based therapeutic strategies.